It is well acknowledged that some of the volatile plant compounds (VPC) naturally present in insect natural habitats alter the perception of their own pheromone when presented individually as a background to pheromone. However, the effects of mixing VPCs as they appear to insects in natural olfactory landscapes are poorly understood. We measured the activity of brain neurons and neurons that detect a sex pheromone component in a moth antenna, while exposed to simple or composite backgrounds of VPCs representative of the odorant variety encountered by this moth. Maps of activities were built using calcium imaging to visualize which brain areas were most affected by VPCs. In the antenna, we observed differences in VPC capacity to elicit firing response that cannot be explained by differences in stimulus intensities because we adjusted concentrations according to volatility. The neuronal network, which reformats the input from antenna neurons in the brain, did not improve pheromone salience. We postulate that moth olfactory system evolved to increase sensitivity and encode fast changes of concentration at some cost for signal extraction. Comparing blends to single compounds indicated that a blend shows the activity of its most active component, VPC salience seems more important than background complexity.Agrotis ipsilon while exposed to simple or composite backgrounds of a panel of VPCs representative of the odorant variety encountered by a moth. Maps of activities were built using calcium imaging to visualize which areas in antennal lobes (AL) were affected by VPCs. We compared the VPC activity and their impact as backgrounds at antenna and AL levels, individually or in blends. At periphery, VPCs showed differences in their capacity to elicit Z7-ORN firing response that cannot be explained by differences in stimulus intensities because we adjusted concentrations according to vapor pressures. The AL neuronal network, which reformats the ORN input, did not improve pheromone salience. We postulate that the AL network evolved to increase sensitivity and to encode for fast changes of pheromone at some cost for signal extraction. Comparing blends to single compounds indicated that a blend shows the activity of its most active component. VPC salience seems to be more important than background complexity.The volatile plant compounds (VPC) alter pheromone perception by insects but mixture effects inside insect olfactory landscapes are poorly understood. We measured the activity of receptor neurons tuned to Z7-12Ac (Z7-ORN), a pheromone component, in the antenna and central neurons in male  Olfactory communication is essential to insects as it is involved in the identification and the location of vital resources such as a food source, a mate, or an oviposition site. Insects have developed an exquisite olfactory sense in terms of sensitivity, specificity, and temporal dynamics. Their olfactory system enables them to discriminate the pheromones they produce, as well as the odors involved in interspecific interactions, such as, for instance, the floral compounds emitted by plants to attract specialist pollinators. Herbivorous species, for instance, can discriminate potential host-plant species based on their volatile emissions . ExchangInsect olfactory systems evolved to deal with such complex olfactory landscapes . Male moSpodoptera littoralis males to deficient or heterospecific pheromone signals in a wind tunnel [Heliothis zea to its pheromone [Agrotis ipsilon [S. littoralis [Heliothis virescens males [Cydia pomonella, also binds the plant volatile pear ester [Odors within the habitat, and most specifically the volatile emissions from host-plants, affect moth behavioral responses to pheromone. Host plant volatiles, for instance, reduce the responses of d tunnel . Plant ad tunnel . Interacheromone . Heptana ipsilon . Howeverttoralis . A backgttoralis . Activitttoralis . Interesttoralis ,32. The ttoralis . There ins males . A sex par ester .H. virescens males was significantly impaired in a concentration-dependent manner after adding single VPCs, their pheromone-guided flight behavior was not influenced by the natural emissions of host-plants in a wind tunnel. Badeke et al [2 and O3, which modifies the amounts of VPCs they release in the atmosphere [The ecological importance of VPC-pheromone interactions in natural conditions was recently questioned  because,ke et al  concludemosphere ,38. Due Agrotis ipsilon (Hufnagel). The black cutworm, A. ipsilon, is a polyphagous and cosmopolitan moth belonging to the Noctuidae family that causes economic losses to many crops around the world. As in most moth species, pheromone communication is crucial for A. ipsilon mating, but males also rely on plant volatiles as food cues [A. ipsilon has become a model species for studying the processing of pheromone and plant signals and their interaction at neuronal and behavioral levels [A. ipsilon consists of three main components: cis-7-dodecenyl acetate (Z7-12:Ac), cis-9-tetradecenyl acetate (Z9-14:Ac), and cis-11-hexadecenyl acetate (Z11-16:Ac) [The present research aimed to investigate the effects of multi-component VPC backgrounds on pheromone perception in the moth ood cues ,40. A. il levels ,32,41. T1-16:Ac) ,43. Thre1-16:Ac) ,45. Prev1-16:Ac) . Only tw1-16:Ac) . A 3D gl1-16:Ac) . We used1-16:Ac) ,29. AccoA. ipsilon adult males were obtained from a laboratory stock. Larvae were fed on an artificial diet [ad libitum. Day of emergence was considered as day-0. Males were aged of four days at experiment time. Experiments were performed during scotophase hours but under day light.ial diet . Pupae wZea mais, one of the host crop plants of A. ipsilon [Artemisia annua, a common weed in maize fields [The pheromone component Z7-12Ac CAS 14959-86-5), was purchased from Pherobank (purity > 99%). Based on the literature, we selected eight VPCs with different chemical structures and physicochemical properties to be representative of the variety of odorants that can be encountered by a male moth searching for a mate in an agricultural landscape in mainland France. The monoterpene linalool, the aromatic heterocyclic indole, and the bicyclic sesquiterpene β-caryophyllene have been identified in constitutive and herbivore-induced emissions of  ipsilon . α-pinen59-86-5,  ipsilon ; it is r ipsilon . (Z)-3-h ipsilon . Eucalype fields . VPCs weThe stimulus delivering device consisted of an ensemble of electrovalves enabling to deliver VPCs from separate sources in the main air stream of a glass tube  whose distal end was positioned 20 mm from the insect antenna, while the pheromone stimuli were delivered through a lateral input . Air wasPheromone stimuli were delivered as air puff (167 mL/min) through a Pasteur pipette containing a piece of filter paper loaded with 10 ng (unless mentioned) of Z7-12:Ac diluted in 1 µL of hexane. Hexane was left to evaporate for 30 s before inserting the pheromone-loaded paper into the pipette. Air passage through the pipette was commanded by an electrovalve . The pipette tip was inserted into a hole on the side of the glass tube, 150 mm upstream of its distal end. VPC sources consisted of 4 mL glass vials containing 1 mL of a single VPC diluted in mineral oil, or mineral oil only (control). The air stream was divided into 8 parallel flows (200 mL/min each) with an airflow divider , each of which directed towards a 3-way electrovalve . Normally opened  and normally closed  exits of the eight valves were connected either to empty vials or to VPC source vials, respectively. All outlets of odorized and non-odorized vials were connected to the proximal end of the glass tube. Thus, valve opening did not modify the total airflow received by the antenna (1.6 L/min). All tubing downstream from the valves was made of Teflon . Vials were connected to the tubing with stainless steel hypodermic needles inserted through a Teflon septum. For delivering single VPCs, the Teflon tubes at vial outlet were directly connected to the main glass tube . VPC mixStimulation sequences consisted of a short pheromone puff delivered in the middle of a 5 s VPC background presentation . For eleA. ipsilon had been already demonstrated [First, we evaluated the effects of single VPCs. Linalool, whose ability to activate the MGC-neurons of nstrated , was usenstrated  based onnstrated   necessary to obtain the desired concentration in the headspace (hC) of a closed, equilibrated source:Then, the effects of binary blends of VPCs and their components alone were tested at 4 concentrations each, while the pheromone dose was kept constant. In order to control precisely the ratios in the background, we measured their air–mineral oil partition coefficients Khl,  and usedhlK were measured by injecting the headspace of closed equilibrated sources containing known concentrations of VPCs into a calibrated GC-FID. When an airflow passes through such a source, the concentration in the headspace drops to a fraction of the initial concentration and then reaches a steady state until the source starts to exhaust. Therefore, odorant concentration delivered from the source is related to the initial concentration inside the closed equilibrated source by a dilution factor that does not depend on the odorant. Assuming that this proportionality remains true for the concentrations delivered on the antenna , we expressed all delivered VPC concentrations relative to an arbitrary unit (AU) where one AU was defined as the molar concentration delivered on the antenna from a source containing 1% linalool in mineral oil. Partition coefficients Binary blends were obtained by opening simultaneously the valves controlling the airflow through the sources containing the single VPCs to be blended while closing a compensation flow. Therefore, if the concentrations of blend components, when tested separately, are noted A and B, the blend was tested at the concentration A + B. The order of presentation of individual VPCs and of their combination was randomized. For each background type, 4 successive stimuli were presented at increasing concentration. A pheromone puff with no background was presented as a control at the beginning of the recording and after the highest concentration of each background type. We verified with a photoionization detector  that the concentration of VPC did not vary depending on whether it was delivered alone or in a multicomponent blend, as well as along successive stimuli separated by 5 or 10 min, with the exception of the very first stimulus . ExperimFinally, we tested blends of 3 or 4 VPCs as backgrounds to the pheromone stimulus. We alternated presentation of these complex backgrounds with backgrounds consisting in a single VPC or a binary blend of its components. Each of these 2 composite backgrounds and the corresponding simple backgrounds were presented twice to each neuron in random order. Control stimuli (pheromone without background) were presented at the beginning of each recording and after each pair of composite and simple background stimuli.2 and restrained in a Styrofoam holder. One antenna was immobilized with adhesive tape. A tungsten electrode was inserted into the antenna to serve as a reference. We targeted the ORNs tuned to the pheromone constituent Z7-12:Ac (Z7-ORN) which are housed in the long trichoid sensilla located along antennal branches. The recording electrode was, therefore, inserted at the base of one of these sensilla. For single sensillum recordings, male moths were briefly anesthetized with CO2 6, MgCl2 2, Hepes 10, Glucose 5 . The preparation was constantly perfused with this solution once the brain capsule was opened. The reference electrode was a silver wire inserted at the rear of the head capsule in contact with brain tissues. The recording electrode was slowly inserted inside the MGC until the appearance of a clear single-unit firing activity. Extracellular recordings from A. ipsilon AL sample only neurons with a large neurite [For extracellular recordings from MGC-neurons, male moths were restrained in a cut pipette tip, leaving the head exposed, and immobilized with dental wax. The head capsule was opened, and the brain exposed by removing all muscles and mouthparts above it. The neurolemma was carefully removed from the antennal lobe in order to allow electrode penetration. The recording electrode was made from a glass micropipette whose tip was manually broken to a diameter of 2 μm and filled with (in mM): NaCl 150, KCl 4, CaCl neurite  so we exMale moths were restrained in a Plexiglas chamber and the head was fixed. The head was opened and muscles and mouthparts removed to gain access to the brain. Then, 20 µL of a dye solution  was bath-applied for at least 1 h, before being washed with Ringer. For recordings, a T.I.L.L. Photonics imaging system  was coupled to an epifluorescent microscope  equipped with a 10× (NA 0.3) water immersion objective. Signals were recorded using a 640 × 480 pixel 12-bit monochrome CCD camera . The acquisition rate was set at 5 frames/s with an acquisition time of 15 ms. Identification of individual glomeruli was done by superposing activity maps using Adobe Photoshop (Version CS2). We chose not to include isoprene in calcium imaging experiments due to the limited number of channels available in the odor stimulation device.0)/F0 (where F0 = reference background). For each glomerulus, the time course of δF/F was calculated by averaging 25 pixels (5 × 5) at the center of each glomerulus.Raw data analysis was done using custom–made software written in IDL  and Visual Basic (Microsoft Excel). After noise filtering and bleaching correction, relative fluorescence changes (δF/F) were calculated as  from the maximum firing frequency reached in TW2. Similarly, the pheromone response was calculated by subtracting meanTW1 from the maximum firing frequency reached within TW4 (maxTW4). We estimated the capacity of neurons to extract the pheromone signal from the background, the pheromone salience, by subtracting pre-pheromone activity level in TW3 from maxTW4. We calculated the maximum firing frequency within four time-windows (TW) covering the successive phases of the two stimuli. The limits of each TW are the valve opening times shifted to consider the travel time of the odorized airflows from the valves to the antenna . TW1 . C), and depends on a maximal response intensity (maxR), a concentration at half maximum (EC50), and Hill’s coefficient (n):For experiments comparing blends to their components we used a permutational MANOVA  under R package vegan ,59 to evmaxR. However, in the case of pheromone response and of pheromone salience, the aim was to model the decrease in response intensity observed in the presence of an agonist background. For that purpose, response intensity or salience in the absence of background could be considered as maxR. Since individual neurons differed in their responsiveness to pheromone, we normalized all observed pheromone response and salience values to the considered neuron’s corresponding maxR:Our response to background data were not appropriate for a fit of Hill’s equation because the saturation was never reached making it impossible to estimate maxR = 1. Subtracting this simplified Hill’s equation from 1 produces a curve that decreases with increasing C, which is empirically appropriate for fitting on the observed decreasing response to pheromone or pheromone salience as function of the background dose:This also allowed to simplify Hill’s equation by setting 50 and n for the Z7-ORN recordings pooled together (n = 16), and for each background type separately, using total background concentration in arbitrary units as C. We compared the dose-responses observed under blended background and under single compound’s background by checking whether the confidence intervals for the fitted parameters did or did not overlap.We fitted Equation (5) using the non-linear regression function nls2  to estimate ECZ7-12:Ac activated a large area in the AL that we identified as the MGC according to its position, similar to previous observations in the same insect species ,61 Figu. All tesWhen pheromone was delivered during the VPC-background, Ca-responses to VPC and to Z7-12:Ac merged to form a single fluorescence-peak . Thus, ip < 0.0001), partially masking the response to Z7-12:Ac. The lowest active dose was 1% . Recordings showed a fast rise of the firing at the background onset, followed by a plateau lasting the time of the linalool presentation. Linalool 1% was chosen as a reference stimulus for further experiments.As the Ca-signal measured with bath applied calcium dye is generally considered as mainly reflecting the sensory input from ORNs, we undertook to record the firing activity of the MGC neurons. To specify at which VPC dose the MGC neurons are expected to respond, we first tested linalool, a compound previously shown to stimulate MGC neurons activity . Overalln = 10) varied according to the VPC . (Z)-3-hexenyl acetate strongly stimulated the MGC-neurons (p < 0.0001). Compared to linalool, responses to (Z)-3-hexenyl acetate were more dynamic, showing a fast initial peak with a short latency (p = 0.0579) due to the variability of responsiveness of MGC-neurons to this VPC (p = 0.0928). Then, we stimulated moth antennae with the other VPCs of the panel, adjusting their concentrations in the source vial according to their volatilities . Respons latency B, follow latency A–C, but this VPC C. Severathis VPC C suggestp < 0.0001), indicating the occurrence of mixture suppression and resulting in an almost complete masking of pheromone (p = 0.0169). The combination of response to VPC background and reduction in response to pheromone resulted in a significant reduction in pheromone salience in the presence of (E)-2-hexenal (p = 0.0061), linalool (p = 0.0003), and (Z)-3-hexenyl acetate (p < 0.0001) backgrounds. Contrary to the Ca-imaging experiments, it was possible to isolate the increase in firing activity in response to the pheromone puff and to measure the pheromone salience. Response to pheromone was dramatically reduced in a (Z)-3-hexenyl acetate background (heromone C. Reductp = 0.0579) when pooling neurons and response to pheromone and pheromone salience were not statically different between eucalyptol and control background. β-caryophyllene, indole, isoprene, and α-pinene neither elicited significant responses nor significantly altered the response to pheromone . However, compared to MGC-neurons, their firing activity decreased more slowly with a longer tail (p = 0.0004). The firing peak in response to pheromone was hardly visible (p < 0.0001) -3-hexenyl acetate background was similar on ORNs as it was on MGC-neurons . Z7-ORNsger tail B. The re visible B and acc 0.0001) C.p = 0.6724), unlike MGC neurons, nor to a (E)-2-hexenal background (p = 0.6709). Response to pheromone and pheromone salience were not altered by linalool  or (E)-2-hexenal . In agreement with its lack of activity in MGC-neurons, no significant effect of α-pinene was observed in Z7-ORNs, either for response to background (p = 0.5447), response to pheromone (p = 0.6244), or pheromone salience (p = 0.8908). The indole background did not activate Z7-ORNs (p = 0.73542), and did not modify response to pheromone (p = 0.8275) and pheromone salience (p = 0.1126).Z7-ORNs did not respond to the linalool background (p = 0.023 for the comparison of the difference in number of spikes in TW4 and TW3). Such inhibitory responses were not observed for (E)-2-hexenal (p = 0.0621), α-pinene (p = 0.254), indole (p = 0.9611), β-caryophyllene (p = 0.1920), and linalool (p = 0.8499). We also noted a significant increase for (Z)-3-hexenyl acetate (p = 0.0068), due to the response to the background.To highlight putative inhibitions, we applied backgrounds as short pulses over a sustained pheromone stimulation . CompareAltogether, Ca-imaging and electrophysiology allowed to identify (Z)-3-hexenyl acetate, linalool, and to a lesser extent (E)-2-hexenal, as Z7-12:Ac agonists with masking activity of the pheromone responses at the peripheral or AL levels. Eucalyptol was postulated to be a weak inhibitor. The other VPCs, indole, α-pinene, β-caryophyllene and isoprene were postulated to be neutral at the tested concentration. In the next step we combined these VPCs in 2 to 4 component blends to determine whether they interacted at the level of Z7-ORNs.We prepared binary blends by combining VPCs having demonstrated contrasted impacts on the pheromone perception and compared their effects on Z7-ORNs to those of their individual components at different doses. To facilitate comparisons between single components and their blends the concentrations delivered to the antenna were expressed in arbitrary units (AU) as defined in the material and methods.p = 0.001) and responses to the background increased with the concentration of (Z)-3-hexenyl acetate (p = 0.939). When the binary blend was presented as a background, neuron activities were not different from those observed under a background of (Z)-3-hexenyl acetate alone (p = 0.262).We first mixed the agonist (Z)-3-hexenyl acetate with α-pinene, which neither stimulated MGC-neurons and Z7-ORNs nor modified their response to pheromone, to determine whether the effect on pheromone response of a VPCs could be antagonized by another VPC. To increase the probability of evidencing an interaction between these two VPCs when blended we doubled the proportion of α-pinene relatively to that of (Z)-3-hexenyl acetate (ratio 2:1). The effects of (Z)-3-hexenyl acetate on Z7-ORNs were clearly dose-dependent . At high concentrations, linalool activated Z7-ORN, confirming a dose-dependent agonist activity at ORN level . Response to the linalool background was apparent only at 4AU . The blend was also more active than (Z)-3-hexenyl acetate alone at the two highest concentrations -3-hexenyl acetate versus blend, background type effect p = 0.001). We then mixed the two agonists (Z)-3-hexenyl acetate and linalool at a 1:1 ratio and compared the blend effects to that of its individual components. Comparison of the activity of Z7-ORNs in response to increasing doses confirmed that linalool was a weaker agonist than (Z)-3-hexenyl acetate (pairwise PERMANOVA (Z)-3-hexenyl acetate versus linalool, background type effect, However, since the global blend concentration was the sum of that of its components we could not determine which type of blend interaction occurred between (Z)-3-hexenyl acetate and linalool. To this end, we modeled the background doses-response to pheromone and pheromone salience curves for each of the three background types . Our modp = 0.444, p = 0.908). Similarly, the 4-component background did not influence neuron firing and response to pheromone any differently from either (Z)-3-hexenyl acetate alone .Finally, we tested the effects of two more complex backgrounds on pheromone detection by Z7-ORNs. A 3-component background was prepared by mixing the agonist (Z)-3-hexenyl acetate with indole and β-caryophyllene, at a ratio of 1:1:0.3. A 4-component background was prepared by mixing the two agonists, (Z)-3-hexenyl acetate and linalool with α-pinene and eucalyptol, at a ratio of 1:1:2:2. The 2-component background obtained by mixing linalool and (Z)-3-hexenyl acetate was included as a reference. All blends and reference backgrounds were presented at (Z)-3-hexenyl acetate concentration = 1 AU. The 3-component background did not influence neuron firing and response to pheromone any differently from (Z)-3-hexenyl acetate alone . This suggests that VPCs can alter the pheromone binding, whether it be by syntopic or allosteric interactions. It makes difficult to predict the impact of one VPC on pheromone detection. Interestingly, we observed large differences between VPCs in the temporal dynamics of the firing responses. A fast response followed by a decrease was observed for (Z)-3-hexenyl acetate, while linalool elicited a delayed increase in firing. Differences in the time-course of odorant concentrations due to air-surface interactions within the system delivering the stimuli have been measured by physical methods in an olfactometer [VPCs differed in their capacity to stimulate Z7-ORNs and such differences cannot be explained by stimulus intensities because we adjusted the source concentrations to vapor pressures. Thus, these differences must be attributed to the binding selectivity of ORs or any other olfactory proteins. Although in the present work the pheromone salience was lower in a background of VPCs activating the Z7-ORNs, other studies revealed that responses to pheromone can also be decreased by VPCs with no intrinsic activity . A clearctometer ,66. Odorctometer . In several moth species the integration of pheromonal signal and host plant odors occurs within the male ALs ,68,69. BVPCs are naturally emitted by plants in complex mixtures. While each VPC might individually be present at very low mixing rates within the atmosphere, the total levels of VPCs over a forest commonly reach hundreds of ppb to peak at nearly one ppm . It is t2 concentration and temperature elevations on plant physiology indicates a fast global increase in their VPC emissions [Manduca sexta the background has important effects on the moth’s ability to locate its host plant [A. ipsilon, will have a greater impact on insect olfactory communication than botanical changes associated to less potent VPCs. It is thus important to better evaluate the impacts of the insect exposure to various odorscapes. Analyses of interactions at the molecular level, will contribute to better predict the risks. We also need quantitative analyses of odorscapes at fine temporal and spatial scales to better estimate the VPC peak concentrations and exposure durations insects experience. Insects have evolved their efficient pheromone communication system in the presence of a complex natural background of VPCs . Howevermissions  and signmissions . This inmissions . In Mandst plant . Interes


It provides biocompatibility and avoids protein adsorption leading to inflammation and rejection of implants. Moreover, hydrogels can be loaded with biologically active compounds. In this field, hyaluronic acid has been largely studied as an additional component since this polysaccharide is naturally present in extracellular matrix. Strategies to direct hydrogelation processes exclusively from the surface using a fully biocompatible approach are rare. Herein we have applied the concept of localized enzyme-assisted self-assembly to direct supramolecular hydrogels in the presence of HA. Based on electronic and fluorescent confocal microscopy, rheological measurements and cell culture investigations, this work highlights the following aspects: ( Supramolecular hydrogels resulting from the self-assembly of low molecular weight hydrogelators (LMWH) in water  give ris11 and IKVAV-Q11 have been also co-assembled leading to β-sheet organization in nanofibers   = 5 mg/mL). We focused our attention on the diameter values of the self-assembled single fibers, called elementary fibers (EF). In order to observe them properly, hydrogels were vortexed and diluted with borax buffer before observation. All calculated values result from the mean of thirty measurements at least, randomly selected on several TEM images. Without HA, the self-assembly of Fmoc-FFY leads to elementary nanofibers with 5.8 ± 0.9 nm diameter. When 2, 5 and 10 mg/mL of HA is present with Fmoc-FFpY, the addition of AP leads to EF of 5.9 ± 1.0 nm, 5.8 ± 0.8 nm and 6.0 ± 1.1 nm respectively  is a tool to get information related to the chirality at the molecular and supramolecular scale. In particular, it allows for identifying patterns in self-assembled architectures. Thus, when AP is added to a Fmoc-FFt 303 nm ,44. In p2 (PEI = poly(ethylene imine); PSS = poly(styrene sulfonate); PAH = poly. This multilayer is capped with a polycationic layer due to PAH, allowing the adsorption of AP, and leading to the enzymatically active thin film PEI/(PSS/PAH)2/AP. This multilayer architecture ensures to be independent of the substrate’s nature. Then, these polymer multilayer films were dipped in borax buffered solution (pH 9.5) of Fmoc-FFpY (5 mg/mL) containing 0, 2, 5 or 10 mg/mL of HA, during 12 h before analysis by cryo-SEM. To observe both the thickness of each hydrogel formed and its internal architecture, each sample was frozen in liquid ethane, then broken and finally freeze-dried. No metallization has been done. In all studied conditions, cryo-SEM images show a full covering of the substrate by the hydrogel layer , as expected. However, it appears some small globules distributed everywhere on the surface. This could be due to Fmoc-FFpY aggregates as already reported for other Fmoc-peptides hydrogelators. When the enzymatically-active multilayer film PEI/(PSS/PAH)2/AP is brought in contact with Fmoc-FFpY solution (5 mg/mL), a dense fibrous network is observed. The presence of HA (2 mg/mL) leads to larger structures that could be formed from the assembly of thin nanofibers in presence of HA. This influence of HA on the internal peptide self-assembled architecture grew up from the surface is confirmed by SEM images taken from surface: kind of veil structures are observed when HA is present (2 or 5 mg/mL) whereas only thinner nanofibers constitute the supramolecular hydrogel in absence of the polysaccharide, as described previously through cryo-SEM studies  were brought in contact with the hydrogels for 4 h at 37 °C. Then, a life/dead fluorescent test was performed to obtain the cell viability values for each hydrogel . In comppY and we have shown that additional polymer components such as HA can be incorporated during the spatially localized hydrogel growth to get hybrid peptide/polymer coatings displaying good cell adhesion and spreading. Thus, HA does not prevent the hydrogelation process and the variation of its proportion allows to modulate the resulting hydrogel thickness. Furthermore, as the HA proportion increased, the storage modulus of the resulting hydrogel decreased, a balance that can be useful to get very soft hydrogels to be employed as essential substrates for nerve cell cultures, for instance. Based on electron and fluorescence microcopy investigations, the incorporation of HA does not change the shape of the peptide-based nanofibers, but it has a strong impact on their ability to collapse together, generating large microstructures. The study of the origin of this interaction between HA and EASA is currently under investigation by our group.The spatial localization of enzymes on polymer multilayer films allows us to direct the peptide self-assembly exclusively where enzymes are located. This LEASA approach is a powerful tool to design micrometer thick supramolecular hydrogel coatings from any kind of substrate using the multilayer film method. Herein, we have used the tripeptide hydrogelator Fmoc-FFY generated in situ from the system AP/Fmoc-FF


Z isomer leads to a strong batochromically-shifted absorption band, allowing for fast isomerization to the metastable E isomer with light in the 500–600 nm region. Theoretical studies of the reaction mechanism reveal the crucial role of the acid–base equilibrium which controls the populations of the protonated and neutral forms of the E isomer. Since the former is thermally stable, while the latter re-isomerizes on a millisecond time scale, we are able to modulate the half-life of ITIs over three orders of magnitude by shifting this equilibrium. Finally, stable bidirectional switching of protonated ITI with green and red light is demonstrated with a half-life in the range of tens of seconds. Altogether, we designed a new type of multi-responsive molecular switch in which protonation red-shifts the activation wavelength by over 100 nm and enables efficient tuning of the half-life in the millisecond–second range.Multi-responsive functional molecules are key for obtaining user-defined control of the properties and functions of chemical and biological systems. In this respect, pH-responsive photochromes, whose switching can be directed with light and acid–base equilibria, have emerged as highly attractive molecular units. The challenge in their design comes from the need to accommodate application-defined boundary conditions for both light- and protonation-responsivity. Here we combine time-resolved spectroscopic studies, on time scales ranging from femtoseconds to seconds, with density functional theory (DFT) calculations to elucidate and apply the acidochromism of a recently designed iminothioindoxyl (ITI) photoswitch. We show that protonation of the thermally stable  E-isomer over three orders of magnitude.Protonation of an ITI molecular photoswitch shifts its absorption to the orange/red part of the spectrum and allows for manipulation of the thermal half-life of the photogenerated  Protonation of molecular photoswitches has important consequences for their spectral properties, isomerization mechanism and half-life of the meta-stable form, and has been recognized as a simple and reversible method for tuning the photochromes for particular applications. These efforts are often inspired by nature, where protonation plays a key role in the switching function of e.g. retinal Schiff bases,10 fluorescent proteins,11 proton pumps,12 and phytochromes.13Due to the outstanding spatiotemporal resolution offered by light delivery, molecular photoswitchestetra-ortho-alkoxy-azobenzene 1  (OPE)-embedded diarylethene 5 as well.18Various types of molecular photoswitches  have beeenzene 1  introducpyrane 4 .17 In th19 while photoisomerization can be employed to change the proton affinity of a molecule in reversible photoacids and bases.20 Examples of the first approach include hemithioindigo 6,21 in which protonation of the strongly electron-donating dimethylamino group changes its electronic character, thus inducing a band shift and turning 6 from a P-type into a T-type switch; similar effects were observed for hydrazone 7.22 The photoacid designs are most often based on modified spiropyrans23 such as compound 3,24 while photoswitchable basicity has been realized with azobenzenes25 and azoheteroarenes.26From an application perspective, the relation between protonation and photoswitching can be exploited in both directions: protonation can be used to modulate the switching process and thereby create multi-responsive systems,27 The ITI photoswitch consists of half a thioindigo and half an azobenzene  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N double bond. This fused photoswitch features both the visible light absorption of thioindigo and the band separation characteristics of azobenzene, resulting in a fully-visible-light photoswitch with a 100 nm band separation between the Z and E isomers. The Z isomer can be switched to the E isomer using light of around 400–450 nm while back-isomerization can be done photochemically  or thermally, with a half-life of tens of milliseconds at room temperature. The small size of ITI would enable its future introduction into functional systems without affecting their overall properties. However, the fast re-isomerization process constitutes a challenge for many applications, as it limits the concentration of the metastable E isomer that can be achieved under irradiation. For example, based on MRI and PET data of mean transit times for blood in the cerebral vasculature,28 Woolley and co-workers suggested15 that the optimal thermal half-life should be in the 0.1–10 s range to provide an optimal spatial resolution. This is an order of magnitude higher than that found for ITI. Furthermore, bathochromically shifting the absorption maximum of ITI would allow switching with non-destructive, low-energy visible/red/NIR light and thus greatly expand the scope of applications. Moreover, it would enable selective ITI operation in complex mixtures with other components absorbing in the UV-violet-blue, and thus offer unique possibilities for the design of orthogonal switching systems.29Here we explore the influence of protonation on iminothioindoxyl (ITI), a new member of the family of visible-light-operated photoswitches.obenzene , which rE–Z re-isomerization is assumed27 to proceed through thermal in-plane inversion of the imine nitrogen atom.30–32 In this process, a linear intermediate is formed, after which the imine relaxes to the thermally favored Z state. With this in mind, we hypothesized that engaging the imine-nitrogen electron pair in a transient covalent bond with a proton would inhibit fast re-isomerization and enhance the thermal stability of the metastable E isomer. Furthermore, inspired by previous reports,15,16 we also expected it to result in a significant bathochromic shift of the absorption bands of both isomers, thereby meeting our second objective.Detailed understanding of the isomerization mechanism enables the tuning of photoswitches through molecular engineering and pathway selection. In the context of ITI, the fast In this work, we present a combined experimental and theoretical study of the photochemistry of protonated ITI switches. Using steady-state and time-resolved spectroscopies on time scales ranging from femtoseconds to seconds in combination with time-dependent density functional theory (TD-DFT) calculations, we unravel the mechanistic and spectral aspects of ITI acidochromism and showcase ITI variants that feature optimal half-lives of several seconds and absorption spectra extending into the 600–700 nm region.8a–d (8a) and ITI derivatives containing either electron-withdrawing  or electron-donating  groups in the para position of the phenyl ring. Importantly, as discussed further in Section S2 of the ESI,33,34To investigate the influence of protonation on the spectral properties of ITI switches, we titrated a range of compounds 8a–d  with tri8a–d . The cho8a–d feature two absorption bands in their UV spectra: a strong one at λ ∼400–450 nm associated with the S0 → S2 electronic transition, and a weak one at λ ∼350 nm which is attributed to the S0 → S3 transition (vide infra).27 Upon addition of TFA to the ITI solutions, we observed the emergence of two new absorption bands: one at roughly the same spectral region as the S0 → S3 transition in the neutral form, and one that is significantly red-shifted and is observed in the 450–550 nm region, tailing to over 600 nm in the case of methoxy-substituted ITI 8d. Intriguingly, the relative intensities of the emerging bands strongly depend on the electronic nature of the substituent at the phenyl ring. For the EWG-substituted system 8b, the higher energy band at λ ∼350 nm dominates the spectrum, but this effect is smaller for unsubstituted ITI 8a and further decreases with the increasing electron donating character of the substituents. For compound 8c, featuring a weakly electron-donating methyl substituent, the bands are almost equal in intensity. The even stronger electron-donating character of the methoxy group in compound 8d results in a pronounced absorbance of the red-shifted band, while in the UV region of the spectrum no increase is observed.Neutral ITIs C1/2, 8b–8a–8c–8d is accompanied by an increased proton affinity. Furthermore, the effects observed upon titration with TFA can be fully reversed by addition of triethylamine (TEA)  . These o9 as a model compound that does not feature the CN double bond. Addition of TFA to a solution of compound 9 in DCM  as was previously observed for azobenzene compounds.14,15Aiming to further exclude the influence of non-specific solvatochromic effects, and to confirm the role of the nitrogen atom as the protonation site in ITI switches, we used the homologous hemithioindigo switch 9 in DCM  resulted8d, which featured the most pronounced absorbance of the red-shifted band, are shown in 8a, 8c) are shown in the ESI  obtained from global analysis of the data recorded for protonated compound 27 The time-resolved spectra of the neutral compounds, as shown in 8d, are dominated by a very intense broad excited-state absorption band (ESA), which, according to the global analysis of the recorded kinetic traces, decays on a very fast 0.38 ps timescale. In line with our previous interpretation, we assume that for neutral samples excitation with visible light populates the bright S2 excited state, which undergoes internal conversion to S1 on an ultrafast timescale. Isomerization then proceeds on a time scale competing with vibrational relaxation in S1. The molecule thus relaxes to the ground state of either the Z or E isomer where vibrational cooling takes place in about 9 ps. The long-living spectral component displays a differential line-shape because of the emergence of a red-shifted positive product band assigned to the E isomer27 , for protonated compounds, the excitation with visible light populates the S1 state, which gains oscillator strength upon protonation. Importantly, ultrafast transient absorption spectra show also for protonated compound 8d the emergence of a positive product band , showing that the observed spectral changes and band separation are consistent throughout the studied range of compounds.According to our calculations  band peaking at about 460 nm and a negative broad band assigned to the convolution of bleaching and stimulated emission. On a fast 0.5 ps timescale, the stimulated emission mostly recovers, while the ESA slightly blue shifts as a result of a fast relaxation process that brings the system out of the Frank–Condon region. During the subsequent evolution, occurring on the 4.6 ps timescale, a positive band peaking at 580 nm emerges, while the ESA band mostly decays. We associate this evolution with the reaching of a conical intersection (CI) from which the molecule relaxes to the Z and E ground states. In the ground state, the system undergoes vibrational relaxation within 9.7 ps. As mentioned above, the long-living spectral component is interpreted in terms of a differential E–Z ground state absorption. The nature of the substituent on the phenyl ring has also an influence on the excited state decay kinetics, but we do not observe a clear distinction among EWG and EDG moieties. Indeed, our measurements show that the slowest kinetics is measured in the case of the unsubstituted compound 8a-H+, for which the ESA band recovers in about 40 ps. Faster dynamics is observed for compound 8c-H+ and a further acceleration is noticed for compound 8d-H+  for the investigation of the key stationary points on the ground state (GS) potential energy surface (PES), and its time-dependent counterpart (TD-DFT) for the analysis of the vertical excitations and excited state (ES) geometry optimizations 37,38 and Turbomole39 (ver. 7.3).To gain deeper insight into the effects of protonation on the geometrical features and the nature of electronic transitions in compounds Z and E forms revealed remarkable variations between protonated and neutral compounds 8. In general, the protonated forms are less distorted than their neutral counterparts  character , is a mixed state with a significant n → π* contribution, where n represents to a large extent the lone pair on the nitrogen atom  → π* transition is no longer possible in protonated Z-8-H+. As a result, the S1 state with predominantly ππ* character becomes bright  is again bright (Z-8(a–d)-H+ series is well reproduced by the calculations /SMD levels bright  and the n bright  and manin bright . It is gulations  and can ulations .E-8-H+ forms the theoretically predicted spectra are in good agreement with experimental observations, providing further support for the adopted theoretical approach. As in the case of Z-8-H+, the two observed bands for E-8-H+ can be attributed to S0 → S1 and S0 → S3 transitions. However, in E-8-H+, the S1 state has a mixed ππ*/nπ* character owing to a more twisted structure  plot for the S0 → S1 transition protonation equilibria as displayed in Next, we evaluated if engaging the nitrogen electron pair in a bond with the proton is a viable strategy to increase the thermal stability of the metastable 1 PES for Z-8a-H+, which excluded ESIPT as well as ES deprotonation, implies that photo-activation of the protonated Z form (step 1) leads – through a CI – to the formation of an E-8-H+ isomer (step 2), which can either thermally back-isomerize (step 3) or deprotonate (step 4). The activation barrier for back-isomerization of E-8a-H+ to Z-8a-H+ determined by the composite DLPNO-CCSD(T)/M06-2X/SMD approach43–45 is ca. 30 kcal mol−1, discarding this pathway for E-8a-H+ as well as for the other derivatives  is readily established, opening an alternative path for the back-isomerization in the deprotonated form (step 5). Indeed, the activation barrier for this step (nitrogen inversion) is about 17 kcal mol−1 for 8a  is shifted to the left, i.e., towards the protonated E-8-H+ form, thus decreasing the population of E-8 available for back-isomerization.The analysis of the S8a–d using nanosecond transient absorption spectroscopy. E-8a–d in DCM after nanosecond laser excitation as a function of the TFA concentration. It is gratifying to find that – in agreement with our model – this half-life is strongly dependent on the TFA concentration, showing an increase by more than two orders of magnitude for ITIs 8a, c and d, and somewhat less for 8b.To further experimentally investigate the validity of this theoretical model, we studied the influence of the acid titration on the kinetics of thermal relaxation for ITIs a priori have expected on the basis of the calculated barriers for the direct back-isomerization of E-8-H+ to Z-8-H+  reaching a high photostationary state as is apparent from the considerable drop in absorbance at λ = 532 nm. At the same time, the increase in absorbance at λ = 652 nm indicates the formation of metastable E-8d-H+. Under the employed experimental conditions (10 °C and stirring) the thermal half-life was found to be on the order of 1 min. Interestingly, we observed a significantly faster reverse switching using irradiation with red light (λ = 652 nm). We thus conclude that protonated ITI 8d-H+ can be operated photochemically in both Z → E and E → Z directions using green/red light.Finally, we have explored the overall switching ability of ITI troscopy . We were46–50 However, their rational design is challenging due to the interplay of multiple stimuli-responses and requires detailed understanding of the underlying mechanistic principles at the molecular level. In this context, and inspired by various natural systems, we have explored here the acidochromism of a newly developed, visible-light responsive ITI photoswitch, focusing on the influence of protonation on its photochemical and functional properties.The application of multi-stimuli responsive molecules offers access to advanced and sophisticated functional materials.Z-ITI leads to the emergence of a strong absorption band which can extend to over 600 nm, and the calculations attributed this band to the S0–S1 transition that becomes bright in protonated Z-ITIs. Irradiation at this band results in very fast (∼10 ps) switching to the metastable E-ITI isomer. Detailed theoretical studies of key stationary points on the excited-state and ground potential energy surfaces revealed that the ground-state acid–base equilibrium between E-8-H+ and E-8 plays a key role in the operation of this switch. As a result of a very high barrier for thermal re-isomerization to the Z-8-H+ isomer, modulation of this acid–base equilibrium has been shown to offer an excellent means to actively tune the recovery time of the system for specific purposes. In fact, ITI switches are in this respect on par with other photochromic systems, in which the lifetime of the photochemically produced species can be tuned over several orders of magnitude.15,21,22,51,52 Importantly, we demonstrated that the protonated ITI 8-H+ can be photochemically operated in both directions using low-energy green and red light, similar to the most advanced visible-light-operated P-type photochromes,21,53–55 albeit the relatively short half-life still remaining a challenge.Spectroscopic investigations revealed that protonation of 56Altogether, protonation of ITI leads to red-shifted absorption maxima and longer half-lives, both of great importance for its applicability. However, in the present experiments, the tunability of the half-life has been limited by the employed solvents and acids. We are therefore presently exploring ITI derivatives able to operate under conditions in which this is no longer a limiting factor, also in aqueous solutions of importance for biological applications. In this context, we also recognize the challenges that stem from using protonation equilibria to reversibly manipulate the system with proton concentration, as it results in formation of side products after repeated cycles of acid and base addition. However, combining ITI switching with photochemical, reversible proton generation using a photoacid, as shown before for hydrazone switches, could offer a clean solution to this problem.14,15,52,57 while featuring an obvious structural similarity to hemithioindigo21,58 photoswitches. This striking observation underlines the intriguing nature of ITI as a truly hybrid photoswitch and will hopefully inspire its future applications in designing visible-light-responsive molecular systems.In a broader perspective, the results presented here demonstrate that ITI shows clear functional similarities to visible-light-responsive azobenzenes,M. W. H. H. and W. S. conceived the project and designed the molecules. All calculations were done by M. M. and A. D. L.; M. W. H. H. performed the synthesis. Nanosecond TA spectroscopy was performed by M. W. H. H., J. F., M. H. and W. J. B., while M. T. and M. D. D. performed the femtosecond TA spectroscopic experiments. Steady state UV-vis experiments were performed by M. W. H. H. and W. S. The manuscript was written by M. M., M. W. H. H., A. D. L., M. D. D., W. J. B. and W. S. The research was supervised by W. S., M. M., W. J. B., M. D. D. and B. L. F. All authors discussed the results and progress in all stages.There are no conflicts to declare.SC-012-D0SC07000A-s001


Nowadays, a larger number of aggressive and corrosive chemical reagents as well as toxic solvents are used to achieve structural modification and cleaning of the final products. These lead to the production of residual, waste chemicals, which are often reactive, cancerogenic, and toxic to the environment. This study shows a new approach to the modification of graphene quantum dots (GQDs) using gamma irradiation where the usage of reagents was avoided. We achieved the incorporation of S and N atoms in the GQD structure by selecting an aqueous solution of L-cysteine as an irradiation medium. GQDs were exposed to gamma-irradiation at doses of 25, 50 and 200 kGy. After irradiation, the optical, structural, and morphological properties, as well as the possibility of their use as an agent in bioimaging and photodynamic therapy, were studied. We measured an enhanced quantum yield of photoluminescence with the highest dose of 25 kGy (21.60%). Both S- and N-functional groups were detected in all gamma-irradiated GQDs: amino, amide, thiol, and thione. Spin trap electron paramagnetic resonance showed that GQDs irradiated with 25 kGy can generate singlet oxygen upon illumination. Bioimaging on HeLa cells showed the best visibility for cells treated with GQDs irradiated with 25 kGy, while cytotoxicity was not detected after treatment of HeLa cells with gamma-irradiated GQDs. Since the discovery of GQDs in 2008 by Ponomarenko et al., they have attracted much attention [Graphene quantum dots (GQDs) are a zero-dimensional (0D) nanomaterial that consists of graphene sheets with π-conjugated spttention . They bettention ,4. The ettention . Chemicattention .GQDs are semiconductors with a lateral size below 100 nm and a tunable band gap, usually between 1 and 4 eV ,8, whileOther properties of GQDs are chemical inertness, low cytotoxicity, biocompatibility and good dispersibility in water ,15,16,17One of the methods for GQDs synthesis is electrochemical oxidation. This simple and low-price procedure is based on oxidation and exfoliation of graphite rods, immersed in the electrolyte solution ,24,25. A2 showed more hydroxyl groups compared to the reaction performed in the presence of only water [2O, NH4OH) can cause the shortening, unbundling and annihilation of single-wall carbon nanotubes (SWCNTs) [2O and NH4OH both mixed with isopropyl alcohol) eliminated C–O groups and increased the fraction of sp2-hybridized carbon atoms in the SWCNTs structure. For the first time, GQDs were gamma-irradiated in 2015 [v/v%), while the applied doses of gamma irradiation were 20, 50, 100 and 200 kGy. This study showed that GQDs’ properties such as diameter, photoluminescence intensity, band gap, and singlet oxygen production differ with the change in the irradiation dose. The main improvements in properties were found in the sample irradiated with a dose of 50 kGy: the average diameter decreased from 24 to 18 nm, the oxygen content increased from 35.9 to 45.1 wt.%, photoluminescence (PL) intensity was 4.5 times higher, and the highest production of singlet oxygen upon the UV illumination was detected [Gamma irradiation is a possible approach to the structural modification of carbon nanomaterials . It is aly water . Jovanov(SWCNTs) . Consequ in 2015 . The meddetected .Until now, there have been reported a few top-down syntheses of N,S-doped GQDs. Ouyang et al. reported top-down hydrothermal synthesis from graphite as the carbon source and thiourea as a heteroatom dopant . The stu2 can be achieved [Here, we reported a one-step functionalization of GQDs by gamma irradiation in the presence of IPA as an oxygen radical scavenger and amino acid L-cysteine as a S, N- heteroatom donor. Before irradiation, samples were purged with Ar gas. Due to the presence of IPA, cysteine and the removal of oxygen by Ar gas, the highest concentration of Hachieved . These cachieved ,42, acidachieved , carbamaachieved , etc., oachieved ,46. Thesv/v%). Then, they were dipped in the electrolyte which was prepared by sonication of a NaOH (3 g) in 96 v/v% ethanol (100 mL). Applied current intensity was 20 mA and a voltage was set at 20 V. After 8 h, the color of dispersion changed from light yellow to dark brown, which indicated the formation of GQDs. After that, ethanol was removed with evaporation at a reduced pressure. Obtained as-synthetized material was redispersed in water in a concentration of 3 mg mL−1 and placed into a dialysis bag with molecular weight cut-off (MWCO) of 3.5 kDa. We monitored the pH values of the dispersion all the time. The dialysis was complete when pH remained stable at a value of 7 even after changing the water. In this way, the residual amount of NaOH was removed from the GQDs solution. Water from dispersion was then evaporated by heating at 80 °C to obtain the powder GQDs which were labeled as pristine Graphene Quantum Dots (p-GQDs).Graphene quantum dots were synthesized by using electrochemical oxidation of graphite electrodes purchased from Ringsdorff-Werke GmbH , with 99.999% purity and a diameter of 3.05 mm. Both electrodes, anode and cathode, were graphite ones. Electrodes were washed with Milli-Q water and ethanol . Then, the mixture was purged with Ar gas for 15 min and exposed to gamma irradiation using Co-60 as an irradiation source. Samples were exposed to different doses of irradiation: 25, 50 and 200 kGy. Irradiated samples were dialyzed in the bags with MWCO of 3.5 kDa and evaporated to dryness. The obtained powders were collected and used for further characterizations. Samples were labeled as GQD-cys-25, GQD-cys-50, and GQD-cys-200 which corresponds to applied irradiation doses of 25, 50 and 200 kGy, respectively.To synthesize modified GQDs, p-GQDs were exposed to gamma irradiation. Samples for irradiation were prepared by sonication of GQDs in Milli-Q water at a concentration of 1 mg mL−1. Measurements were conducted in the air environment, at room temperature.Absorption measurements were recorded on a GBC Cintra 6 spectrophotometer  using quartz cell with 1 cm path length and 4 mL volume. UV–Vis spectra were recorded from 200 to 800 nm. For these measurements, GQDs were sonicated in demineralized water for 30 min. The concentration of produced dispersion was 0.25 mg mL−1. Excitation wavelengths for obtaining PL spectra were in the range of 300–400 nm. All measurements were conducted in the air environment at room temperature using a quartz cuvette with a path length of 1 cm and a volume of 4 mL. Fluorescence quantum yields (QY) were calculated using the Equation (1):Photoluminescence spectra (PL) were recorded on a HORIBA Jobin Yvon FluoroMax-4 spectrometer . All of the GQD samples were dispersed in methanol at a concentration of 0.025 mg mL−1. Aqueous dispersions of GQDs in a concentration of 0.25 mg mL−1 were sonicated for 30 min and deposited on mica substrate by spin-coating at 3500 rpm for 1 min. Gwyddion software was used to analyze the lateral size and height of the GQDs. For diameter distribution histograms, we analyzed around 1000 particles, and for height distribution histograms we analyzed more than 100 particles.Atomic Force Microscopy (AFM) measurements were carried out using a Quesant microscope , which was operating in a tapping mode. For non-contact high-frequency applications, we used a rotated monolithic silicon probe Q-WM300, with standard silicon tips  and a force constant of 40 N mThe particle size distribution was determined using the dynamic light scattering (DLS) technique . This system was equipped with a 633 nm helium–neon laser as a light source and measured the particle size at a scattering angle of 173°. All measurements were conducted at a controlled temperature of 20 °C and fixed positions (4.65 nm) with an automatic attenuator.Scanning electron microscopy (SEM) with energy-dispersive X-ray (EDS) spectroscopy was conducted using an FEI ESEM Quanta 200 microscope . Samples p-GQDs, GQDs-cys-25 and GQDs-cys-200 were prepared by dissolving the pristine powders in ethanol; the so-prepared solutions were drop-casted onto clean Si-substrates (10 mm × 10 mm), and finally the samples were dried under vacuum overnight. For SEM imaging, only sample GQDs-cys-50 was deposited onto Al support using the same procedure. Samples were scanned at the SEM microscope without any further preparation , and using the “low vacuum mode”. Energy-dispersive X-ray (EDS) measurements were obtained focusing the analysis over an area of ca. 230 × 200 microns. All samples were deposited on Si support. Data analysis was conducted using the EDAX Genesis EDS microanalysis software .−1. All of the GQDs samples were in powder form.Fourier-Transform Infrared (FTIR) spectra were acquired using the Thermo Scientific Nicolet 6700 FTIR instrument  in attenuated total reflection (ATR) mode. The spectral range was from 900 to 4000 cm1O2 was measured in GQDs before and after gamma irradiation, where air saturated dispersions were mixed with TEMP and exposed to light. The spin trap method is based on the chemical reaction between TEMP and 1O2, which forms a stable radical adduct, oxyl (TEMP-1O2 or TEMPO). TEMPO shows a triplet signal in the EPR spectrum. To investigate the ability of GQDs to produce singlet oxygen under illumination, samples were dispersed in ethanol in a final concentration of 0.2 wt.%. Then, TEMP was added in a concentration of 30 mM and mixtures were purged with air. These mixtures were exposed to blue light (λ = 360–400 nm) for 100 min. EPR spectra were recorded before and during light exposure. Additionally, GQDs were incubated with TEMPO (3 mM) and after illumination in the same condition, EPR was recorded.The electron paramagnetic resonance (EPR) spectra were recorded using a Bruker BioSpin ELEXSYS-II E540 EPR spectrometer with the following experimental parameters: microwave frequency 9.85 GHz (X-band), microwave power 10 mW, modulation amplitude 1 G, modulation frequency 100 kHz and a sweep time of 60 s. 2,2,6,6-tetramethylpiperidine (TEMP) was used as a spin trap agent. Generation of −1. Untreated HeLa cells were used as control. After 24 h of incubation, the medium was removed, cells were washed three times in PBS and subjected to optical imaging. Cellular uptake of p-GQDs, GQD-cys-25, GQD-cys-50, and GQD-cys-200 was monitored using Olympus BX51 fluorescence microscope Olympus  with Spectrum Aqua filter. Images were captured at 10x and 20x objectives and analyzed using the CytoVision 3.1 software .Aqueous solutions of p-GQDs, GQD-cys-25, GQD-cys-50, and GQD-cys-200 were mixed with cell culture media and added to the cells in the final concentration of 100 µg mL4 cells/well in 96-well plates and allowed to attach overnight. The following day, cells were treated with GQDs  at concentrations of 1, 10, 25, 50, and 100 μg mL−1 for 24 and 48 h. At the indicated time points, medium was removed, cells were washed three times with phosphate-buffered saline and cell viability was determined using MTT assay. Cells were incubated for 1 h in MTT solution, 0.5 mg mL−1 cell culture media, , formed formazan crystals were dissolved in DMSO (dimethyl sulfoxide)  and absorbance was measured at 540 nm using an Epoch Microplate Spectrophotometer . All experiments were performed in triplicates, repeated at least three times. The relative viability of the treated cells was calculated as a percentage of the vehicle control (HeLa cells treated with methanol) set to 100%. Data were analyzed using SPSS Statistics 28 . Due to the technical issues we only obtained data from one treatment with GQDs-cys-50 at concentration 100 μg mL−1 at 24 h time point. Accordingly the mean, standard deviation and statistical significance were not calculated for this point.For -2,5-Diphenyltetrazolium Bromide) cell viability assay (MTT) assay, HeLa cells were seeded at concentration 1 × 10GQDs are produced using an electrochemical approach. Two graphite rods were immersed in 3% NaOH in ethanol and the current was applied. After 8 h, the dispersion with NaOH, ethanol and GQDs was collected. To isolate dots, ethanol was removed by evaporation, while NaOH was eliminated in the process of dialysis. To achieve a one-step modification of GQDs, they were dispersed in a water–IPA mixture and L-cysteine was added. This mixture was purged with Ar and irradiated with doses from 25 to 200 kGy. After isolation in the process of dialysis, gamma-irradiated GQDs were characterized and these results are presented in the following text.The optical properties of pristine and gamma-irradiated GQDs were determined by UV–Vis spectroscopy and the obtained spectra are presented in As we can see, all GQDs samples showed typical absorption peaks in the UV region. For p-GQDs, the main absorption band at around 200 nm can be attributed to π–π* transitions from aromatic the C–C bond ,47. In tIn Colloidal dispersions of GQDs showed a broad PL emission . The cenPL emission is associated with Cπ*→Cπ, Nπ*→Cπ and Oπ*→Cπ transitions for N-doped GQDs . Due to Photoluminescence QYs of gamma-irradiated GQDs were calculated using Rhodamine B as a reference and these results are presented in The morphology, height and diameter distribution of p- and gamma-irradiated GQDs were measured with an AFM microscope. The obtained AFM images and histograms of the height and diameter distribution are shown in All images show well-dispersed, uniform, round-shaped particles without large agglomerates. From the height distribution histogram of all samples, it can be seen that the height of GQDs was between 0.5 and 2.5 nm. Since the thickness of one graphene layer measured by AFM was between 0.7 and 1 nm , it can The effects of surface functionalization on the size distribution in gamma-irradiated GQDs were investigated by using DLS. The obtained number-weighted average diameter distribution function is presented in The results show that both GQD-cys-25 and GQD-cys-50 had a similar shape of the diameter distribution function, while GQD-cys-200 was strongly right-skewed, with a heavy right tail. The average hydrodynamic diameter was approximately 23, 24 and 27 nm for GQD-cys-25, GQD-cys-50 and GQD-cys-200, respectively d. The foThe surface morphology and chemical composition of irradiated GQD-cys samples were determined by SEM-EDS analysis. The obtained results are presented in From SEM images a,c,d,g, To investigate the structure and identify functional groups on the surface of GQD-cys, an FTIR analysis was employed. The obtained results are presented in −1 is attributed to the stretching vibration of OH groups [−1 which corresponds to N–H stretches. These results confirmed the presence of N atoms on the surface of GQD-cys. Three low-intensity bands at around 2970 cm−1, 2930 cm−1 and 2877 cm−1 originated from vibrations of sp2- and sp3-CH groups [−1, found only in the gamma-irradiated FTIR spectra, originated from the vibration of the S–H bond [−1 are related to the background CO2.The strong and broad absorption band at 3421 cmH groups . Unlike H groups . Next, tS–H bond ,56. The −1 and 1706 cm−1 stemmed from C=O stretching vibrations in COOH and the amid-carbonyl (–NH–CO–) group, respectively [−1 also proves that the aromatic structure of GQD-cys was preserved after gamma irradiation [−1 and 1630 cm−1 stems from C=C and C=O stretching vibrations [−1 was present in all samples, and it is the result of C–OH bending vibrations [−1 appeared only in gamma-irradiated samples and it is assigned to the C=S stretching vibrations from the thiocarbonyl groups [−1 could be seen in all analyzed samples, and it stemmed from the vibration of C–O bonds.The broad and characteristic band for GQD-cys with peaked at approximately 1630 cmectively ,57. The adiation . For p-Gbrations ,58. The brations . The intFTIR spectroscopy confirmed the doping of gamma-irradiated GQDs with S- atoms as it was observed by SEM-EDS spectroscopy. Additionally, the successful incorporation of N- atoms in the structure of GQDs was detected. These results proved that gamma irradiation in the presence of L-cysteine and IPA in a reduction atmosphere leads to S,N- doping of GQDs.1O2 trap. In the reaction with singlet oxygen, the EPR silent TEMP is converted to the EPR-active TEMPO radical, characterized by an isotropic three-line signal [1O2 to a greater extent than GQD-cys-25.EPR spectroscopy was used to investigate the ability of GQDs to produce singlet oxygen upon light illumination 360–400 nm), using TEMP as a selective –400 nm, The cellular uptake of p-GQDs, GQD-cys-25, GQD-cys-50 and GQDs-cys-200 into HeLa cells was analyzed using fluorescent microscopy and results are presented in −1) as well as exposure time (24–48 h) on the cell viability were studied. All measurements were conducted in dark, to avoid photo-induced toxicity. The results of the MTT assay showed that p-GQDs were not considered toxic toward HeLa cells at concentrations up to 100 μg mL−1 . SEM-EDS analysis indicated that S atoms were present in GQDs. FTIR spectroscopy proved that, apart from S groups (C–SH and C=S), GQDs have N groups such as amino and amide groups. Gamma irradiation caused a small increase in the GQD diameter  compared to p-GQDs (14 nm), dots preserved a round shape, they were homogenously dispersed and no agglomerates were observed. The height of dots was lowered and the highest fraction of single and double-layered dots  was detected in the GQDs-cys-200 sample. The ability of dots to generate singlet oxygen upon light illumination was detected only for GQDs-cys-25 and at the same time this sample showed the best visibility of HeLa cells on a fluorescence microscope. According to the MTT assay, gamma-irradiated GQDs were not toxic toward HeLa cells after treating them with concentrations from 1 to 100 μg mL−1 during 24 and 48 h. These results led to the conclusion that GQDs-cys-25 is a good candidate for both photodynamic therapy and bioimaging. To further improve the structure of irradiated and modified GQDs for use in biomedicine, our future experiments will be based on this and related studies.In this study, we exposed a water dispersion of GQDs mixed with L-cysteine (2 wt.%), and IPA 1


N-diazeniumdiolate (NONOate) as NO donor molecules, using an electrospinning system. We evaluated in vitro NO release kinetics, along with antimicrobial effects and cytotoxicity in microorganisms and human cell culture models. We also studied the long-term stability of NONOates in NO-fibers over 12 months. We demonstrated that the NO-fibers could release NO over 24 h, and showed inhibition of the growth of Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA), without causing cytotoxicity in human cells. NO-fibers were able to store NONOates for over 12 months at room temperature. This study presents the development of NO-fibers, and the feasibility of NO-fibers to efficiently store and deliver NO, which can be further developed as a bandage.Nitric oxide (NO) is a highly reactive gas molecule, exhibiting antimicrobial properties. Because of its reactive nature, it is challenging to store and deliver NO efficiently as a therapeutic agent. The objective of this study was to develop NO-releasing polymeric fibers (NO-fibers), as an effective delivery platform for NO. NO-fibers were fabricated with biopolymer solutions of polyvinyl pyrrolidone (PVP) and ethylcellulose (EC), and derivatives of  The delivery of gases has attracted great attention from researchers over the years, with its potential applications ranging from medicine to the environment and energy storage . The useNO is a free radical gas molecule that is endogenously produced in mammals by the enzyme nitric oxide synthase. NO plays a key role in the innate immune response against foreign organisms, including bacteria ,6,7. TheN-diazeniumdiolates (NONOate), offers a promising solution to this problem, as these donor molecules are able to stabilize chemically, and subsequently release NO when required [The development of small-molecule NO donors, such as required . Each NOrequired . Additiorequired . Althougrequired .For this reason, the use of polymers is believed to be an effective method in harnessing the antimicrobial properties of NO, since the treatment of topical infections involves the local delivery of NO to injured areas . TherefoPseudomonas aeruginosa (PAO1 strain) and methicillin-resistant Staphylococcus aureus  were generously provided by Dr. Anthony Maresso at Baylor College of Medicine . Human microvascular endothelial cells and human dermal fibroblasts, cell culture media and supplements, mini dialysis tubes, and alamarBlue cell viability assay kits were purchased from Thermo Fisher Scientific .All chemicals used in this study, including polyvinylpyrrolidone  and ethylcellulose , were purchased from Sigma-Aldrich  unless noted otherwise. Proline (PROLI)–NONOates, dipropylenetriamine (DPTA)–NONOates, and diethylenetriamine (DETA)–NONOates, and nitrate/nitrite colorimetric assay kits were purchased from Cayman Chemical Company . For the electrospinning process, we first prepared polymer solutions containing NONOates. Briefly, 10% PVP and 8.5% EC solutions were prepared by dissolving PVP or EC in ethyl alcohol with mechanical stirring and heating at 60 °C. Ten milligrams of NONOates, PROLI–, DPTA– and DETA–NONOates were individually dissolved in sodium hydroxide  solution and then added to 20 mL of PVP or EC solution to yield 2.3–3 mM NONOate concentrations. The resulting solutions were vortexed sufficiently and then transferred into a cartridge to fabricate NO-fibers.A desktop electrospinning system  is a unique system where the collector is placed above the emitter. This setup prevents accidental spoilage of nanofiber sheets because of the dripping polymer solutions on the collected nanofibers during the spinning process. The tip of the syringe containing the biopolymer solution was connected to a single jet emitter, where the high electric voltage was applied. The polymer solution containing NONOates was injected at a rate of 30 µL per minute and emitted through the injector tip (19 gauge) upon applying a high voltage (10–15 kV). The emitted fibers were deposited on a grounded rotating collector at 1000 rpm. The distance between the emitter and the collector was maintained at 10 cm. The collected nanofibers containing NONOates, NO-fibers, were carefully separated from the collector and then cut into circular samples 8 mm in diameter for in vitro evaluations. Six-to-eight NO-fibers of each type were kept in air-tight containers to prevent moisture exposure until further evaluations.For scanning electron microscope (SEM) imaging, we used a field emission SEM . NO-fibers were cut into small pieces, 2 × 2 mm, then placed on carbon tape mounted on an aluminum stub. The samples were sputter-coated with gold/palladium at 4 nm thickness . Images were obtained using a secondary electron detector, 5–20 kV, and at a 9 mm working distance. Diameters of NO-fibers were measured from obtained images using ImageJ software . At least 12 fibers were measured from three images.A release study was performed in triplicate on each fabricated NO-fiber in order to evaluate NO release kinetics. To trigger the NO release from NO-fibers, each NO-fiber sample was placed in a mini dialysis tube (MWCO: 2000Da) with 500 µL of phosphate-buffered saline (PBS). The mini dialysis tube was then placed in a 5 mL centrifuge tube also containing 500 µL of PBS. Samples were maintained at 37 °C in an orbital shaker . PBS was collected and replaced with an equivalent amount of fresh medium at the following times: 5 min, 10 min, 30 min, 60 min, 90 min, 2 h, 24 h. Collected samples were evaluated using a nitrite colorimetric assay. Absorbance was measured at 540 nm via a microplate reader . Absorbance values of standard wells were plotted to obtain a nitrite standard curve.g for 10 min to collect the supernatant. Samples were then analyzed using a nitrite colorimetric assay to determine the total NO content. The NO release was determined as follows:We determined the total NO content from the NO-fibers. Each NO-fiber was dissolved in 250 µL of EtOH, followed by 250 µL of PBS. For EC NO-fibers, solutions were centrifuged at 13,000× Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) stocks were kept frozen at −80 °C until required for experiments. Two days before the experiment, each strain was streaked on a Luria broth (LB) agar plate and incubated at 37 °C overnight. A single colony was picked and inoculated in LB broth. MRSA and P. aeruginosa were cultured as planktonic cells in LB broth at 37 °C overnight. Bacteria were then diluted in LB broth (1:100) until the optical density (OD) at 600 nm reached 0.3–0.6 . The bacterial culture was then appropriately diluted to 1 × 107 CFU/mL in LB broth then the culture was transferred to each well in a 96-well plate. To each well, one NO-fiber sample was added. The plate was incubated at 37 °C, and the OD at 600 nm was measured for 24 h at 15-min intervals . Fresh LB and LB with one NO-fiber sample were added to wells as controls to determine possible interference; the presence of NO-fibers did not affect OD as NO-fibers became transparent upon hydration. The growth curve was plotted over 24 h, and the final OD values at 24 h were used to determine the viability. The percent viability was determined as follows:2 in medium 106 supplemented with low-serum growth supplement and medium 131 supplemented with microvascular growth supplement, respectively. Cells were cultured as monolayers in tissue-culture treated 75 cm2 flasks. Once confluency levels reached 85%, cells were trypsinized and passaged. HDFs used in the evaluation were between passages 3 and 6. HMVECs used in the evaluation were between passages 5 and 7.The cytotoxicity of NO-fibers was evaluated in human dermal fibroblast cells (HDFs) and human microvascular endothelial cells (HMVECs). HDFs and HMVECs were maintained at 37 °C with 5% CO4 cells per well). Once cells were added, the plate was incubated overnight in order for cells to adhere to the surface of the wells. One NO-fiber sample was added to each well using a Transwell insert, and the plate was incubated for 24 h. Transwell inserts were used to ensure complete removal of NO-fibers before performing viability assay to prevent interference with absorbance values. After 24 h, NO-fibers were removed from wells, and cell viability was evaluated via alamarBlue cell viability assay. Absorbance was measured at 570 nm via a microplate reader . Wells without films were used as control.HDFs/HMVECs were seeded in each well of a 24-well plate . Moreover, p values < 0.05 were considered to be statistically significant.All experiments were performed with at least three samples. Data are shown as mean ± standard deviation. Statistical analyses were performed by an unpaired, two-tailed Student We used electrospinning technology to fabricate NO-releasing polymer-based nanofibers. Electrospinning is a highly reproducible process that produces polymeric fibers ranging from a few nanometers to micrometers, and with a high surface-to-volume ratio. Furthermore, it allows for easy control of the mechanical properties of the electrospun fibers, such as porosity, which can be easily controlled by manipulating various processing parameters. Specifically, the physical properties of the fibers can be adjusted by altering the polymer concentration, the voltage, and the speed of solution injection.For the successful delivery of NO, the selection of an appropriate biopolymer and NO donor molecule was crucial. The ideal candidate for the biopolymer needs to be highly biocompatible, yield a soft, yet easy-to-handle, final product, and prevent premature NO release. To evaluate the suitable biopolymers as a NO delivery system, we selected PVP and EC as biopolymers to fabricate NO-fibers. PVP and EC are well-characterized synthetic biopolymers that are widely used in biomedical and pharmaceutical research, as well as nanofiber fabrications. PVP has been used as a plasma volume expander and can also be found in personal hygiene products and pharmaceutical applications . EC is aNONOates, along with S-nitrosothiols, are commonly used NO donor molecules, to overcome the limitation of gaseous NO delivery. For S-nitrosothiol molecules, NO is bound to a thiol group and is released upon bond cleavage in the presence of a trigger, specifically metal ions, ascorbate, or light. Thus, while S-nitrosothiol can release NO in a physiological environment, it can only do so under specific conditions ,17,18. MThe current polymeric delivery vehicles for NONOates commonly involve microparticles or nanoparticles, due to their customizability, which allows for distinct release kinetics and targeted delivery . The prePolymeric fibers were formed, as the solvent evaporated when the high voltage was applied A. The emIn our study, we investigated the effect of biopolymers and NO donors on NO release, by fabricating NO-fibers using two different biopolymers and three types of NONOates. We evaluated the in vitro NO release from NO-fibers in PBS at 37 °C to simulate the physiological temperature. Additionally, the NO release is temperature-dependent ,11. The On the other hand, NO-fibers fabricated with EC exhibited immediate NO release within the first 30 min, and no detectable or minuscule amount of NO was released after 2 h. This was attributed to the insolubility of EC in aqueous solvents; NONOates present on the surface of EC NO-fibers instantly released NO when exposed to PBS. For NONOates encapsulated in EC fibers, the water molecules were not able to reach the NONOates and trigger NO release, since the EC NO-fibers did not disintegrate in PBS, due to EC’s hydrophobic nature.We then determined the total NO content from each NO-fiber using the colorimetric assay kit. To understand the effect of biopolymers on release kinetics, we prepared NO-fibers with the same amount of NONOates. We first dissolved both PVP and EC NO-fibers in EtOH, then added PBS to release NO from NONOates. Then, we immediately measured the total NO content by the colorimetric assay. The total NO contents obtained for PROLI NO-fibers were 1.6 ± 0.07 µmol/mL for PVP and 2.8 ± 0.04 µmol/mL for EC. For DPTA NO-fibers, the total NO contents were 4.6 ± 0.3 µmol/mL and 6.7 ± 0.2 µmol/mL for PVP and EC, respectively. The total NO contents were 4.8 ± 0.25 µmol/mL for DETA-PVP NO-fibers and 8.1 ± 0.9 µmol/mL for DETA-EC NO-fibers. The NO encapsulation efficiency was higher in EC NO-fibers than PVP NO-fibers for each NONOate . InteresIn addition to the effect of biopolymers, we evaluated the effect of various NONOates on the NO release kinetics. For our study, we evaluated the effect of different NONOates on in vitro release . Three NPseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). Planktonic bacteria were used for this study, since they are commonly used to evaluate the antimicrobial efficacy against soft- tissue wound infections [P. aeruginosa and MRSA were selected because of their high prevalence among wound infections, as well as their ability to develop resistance to available treatments [P. aeruginosa and MRSA infections, given the rise in antimicrobial resistance among these pathogens.Once we determined the NO release kinetics of each NO-fiber, we then studied the antimicrobial activity of NO-fibers in vitro. After treating with NO-fibers, we evaluated the toxicity on planktonic cultures of two microorganisms, fections . We measeatments ,28. WhilP. aeruginosa was more susceptible to the treatment of NO-fibers than MRSA, as measured in OD at 600 nm. Such differences in sensitivity were expected, since Gram-negative bacteria are more susceptible to antimicrobial agents than Gram-positive bacteria. These differences can be attributed to differences in the cell wall thickness between the two types of bacteria [P. aeruginosa has a considerably thinner cell wall in comparison to Gram-positive MRSA. Thus, the ability of NO to penetrate the cell wall and enter the bacterial cell would presumably be easier for P. aeruginosa than MRSA. Consequently, P. aeruginosa should be more susceptible to NO in comparison to MRSA, which was confirmed by antimicrobial studies.Overall, bacteria . Gram-neP. aeruginosa, which was measured as optical density at 600 nm, compared to untreated P. aeruginosa , and human dermal fibroblast cells (HDF) . We treaThe toxicity of NONOates is not fully established, even though NONOates have been used in human studies as a treatment for acute respiratory distress syndrome ,35. AnotPolymeric delivery vehicles have been shown to overcome any concerns of NONOate toxicity towards human cells, as NONOate incorporating polymeric delivery vehicles showed no toxicity towards human fibroblast cells at concentrations necessary for antimicrobial activity . The bioWe selected HMVECs and HDFs for the evaluation of toxicity, due to the prominent role of each cell type in wound healing ,38. SpecWe evaluated the long-term stability of NONOates in NO-fibers by analyzing the total NO content. NO-fibers in a dry state were stored in sealed containers at room temperature for 12 months before the analyses. Using the colorimetric assay, we measured the total content of NO, and the obtained values were compared to the initial total content of NO (Equation (3)). The evaluation and comparison of NO content showed that NO-fibers retained at least 65% of NO-releasing NONOates after 12 months of storage . Among PAs previously mentioned, the high reactivity of NONOates with moisture is a challenge in the storage and delivery of NO. Therefore, the use of biopolymers was advantageous to stabilize NONOates, not only to prevent the premature NO release, but also to protect NONOates from moisture in the atmosphere. These results showed that the NO-fiber serves as a suitable storage platform for NONOates, even at room temperature.P. aeruginosa and MRSA in vitro, and showed minimal toxicity to HDFs and HMVECs. Although further studies to evaluate in vitro antibiofilm effects of NO-fibers and the antimicrobial effect on skin infection animal models are warranted, we anticipate that NO-fibers will be an effective therapy for treating skin infections involving antibiotic-resistant organisms. NO-fiber application requires minimal effort and allows for infrequent dosing, both of which would be extremely useful for physicians when managing acute skin infections.In this study, we demonstrated the feasibility of the electrospinning process to fabricate NO-releasing polymeric nanofibers. NO-fibers were fabricated from biopolymer solutions of PVP and EC, and the following three different NONOate derivatives: PROLI–, DPTA–, and DETA–NONOate. We showed that electrospun NO-fibers were effective NO delivery platforms, as they prevented the premature release of NO, and released NO over 24 h in vitro. In addition, the release kinetics of NO-fibers were found to be polymer- and NONOate-dependent. We also studied the long-term stability of NONOates in NO-fibers over 12 months. All the NO-fibers retained at least 60% of their original NO content, thus demonstrating the capability of NO-fibers to effectively store NO. Moreover, NO-fibers inhibited the growth of


Moreover, a marginal trend towards significance  was observed between the IL-31 serum level and itch intensity. Our study supports earlier findings on the extended role of IL-31 in the development of CKD-aP.Chronic-kidney-disease-associated pruritus (CKD-aP) is one of the most common and burdensome dermatological symptoms affecting patients undergoing dialysis, and its etiopathogenesis has still not been fully discovered. This study was designed to investigate the possible contribution of interleukin-31 (IL-31) to the pathogenesis of itch in patients undergoing maintenance hemodialysis (HD). We evaluated the serum level of IL-31 in HD patients with pruritus, in HD patients without pruritus and in healthy controls, as well as its correlation to the severity of itch. The study enrolled 175 adult subjects. The participants were divided into three groups. Group A included 64 patients on maintenance HD with CKD-aP, Group B included 62 patients on maintenance HD not reporting CKD-aP and Group C included 49 healthy controls. Pruritus severity was assessed using the Numerical Rating Scale (NRS), and the serum levels of IL-31 were measured. The results showed that the IL-31 serum level was significantly higher in the itchy group ( Chronic-kidney-disease-associated pruritus (CKD-aP) is one of the most common and burdensome dermatological symptoms affecting patients undergoing dialysis. The prevalence of CKD-aP has been very variable over the years, yet according to the most comprehensive observational study of hemodialysis (HD) that included patients from 12 different countries, approximately 40% of patients with end-stage renal disease (ESRD) report moderate-to-severe pruritus . Despiteβ and -6 [Reduced renal function in patients with chronic kidney disease (CKD) precludes the proper elimination of various metabolites, which are known as UTs. Recently, more and more studies indicate the key role of UTs in the progression of CKD and its wide range of severe complications. In the list of medium molecules classified as UTs, a few interleukins can be found, namely interleukin-10, -18, -1β and -6 ,8.Interleukin-31 (IL-31) is a T-cell-derived cytokine that takes part in the symptomatology of pruritus, and both IL-31 and its receptor have become potential therapeutic targets for a range of pruritic disorders . It was p < 0.001) in comparison to the patients free from pruritus. Moreover, there was a significant difference (p < 0.001) in IL-31 serum levels between HD patients with and without pruritus and healthy controls .In the group of HD patients suffering from CKD-aP, 50% were males, and the average age was 61.1 ± 15.9 years. The mean NRS score was 4.9 ± 2.2 points. According to the NRS cut-offs, mild pruritus was reported in 14.5% of the cases, moderate in 59.7%, severe in 22.6% and very severe in 3.2%. The mean UP-Dial score was 14.2 ± 9.8 points. In 58% of the CKD-aP patients, itch interfered with their sleep, and only 29% did not report the influence of itch on the following activities: work or study, social interaction, mood or any sexual activities. The mean serum level of IL-31 was 679.9 ± 1112.3 pg/mL in a group of HD patients reporting CKD-aP, 176.1 ± 290.7 pg/mL in a group of HD patients not suffering from CKD-aP and 57.3 ± 65.1 pg/mL in a group of healthy controls . The IL-p = 0.058) was observed between the IL-31 serum level and the worst itch intensity during the last 3 days as assessed by the NRS . Additionally, each domain of the UP-Dial showed a significant correlation with the NRS scores (detailed data not shown). However, the correlation between the serum level of the IL-31 and the UP-Dial total score was not statistically significant.The UP-Dial questionnaire total score strongly correlated with the NRS . The reason for this discrepancy may be that the present study did not have a big enough study group. Oweis et al. [Otherwise, Ko et al.  additions et al.  also fais et al. .Despite some inaccuracies in the recently carried-out research, the trend of the higher levels of IL-31 in patients experiencing CKD-aP is noticeable. These findings indicate a possible role of the dysregulation of the immune system in the pathogenesis of CKD-aP in HD patients. Kimmel et al.  stronglyIL-31 is a novel cytokine that has a proven role in the pathogenesis of skin inflammation, airway hypersensitivity and in a range of pruritic diseases . StudiesMoreover, therapies targeting IL-31 reduced itch successfully. Grimstad et al.  demonstrThis study showed some limitations. We did not investigate the possible contribution of other inflammatory interleukins in CKD-aP etiopathogenesis. Further studies in this area are necessary. Moreover, the possible correlation between itch intensity and the removal of the IL-31 molecule during the dialysis session should be estimated. The IL-31 with the size of 18 kDa is classified as a middle molecule, and its clearance using high-flux membranes is low . TherefoCKD-aP is a pervasive and multifactorial condition strongly affecting patients’ quality of life. Its etiopathogenesis involves multiple mediators and remains not fully understood. This study revealed a significantly higher serum level of IL-31 in HD patients with CKD-aP versus HD patients without CKD-aP. This tendency supports earlier findings about the extended role of this interleukin in the development of CKD-aP. It is hoped that further research targeting this cytokine may bring more information about the immunological aspect of CKD-aP etiopathogenesis and possible effective and innovative management options for HD patients with CKD-aP.This study enrolled 175 adult subjects of which 85 (48.6%) were females and 90 (51.4%) were males. The mean age of the study population was 58.46 ± 15.8 years. The participants were divided into three groups. Group A included 64 patients with ESRD on maintenance HD with CKD-aP, Group B included 62 patients with ESRD on maintenance HD not reporting CKD-aP and Group C included 49 healthy controls who corresponded with age and gender to the patients from the study group and had no history of any pruritic or systemic diseases. The exclusion criteria for this study was as follows: primary skin disorders, other itchy skin diseases, psychiatric disorder or communication problems, age under 18 years, antipruritic therapy and patients’ refusal. All participants underwent a physical and dermatological examination. Medical history, including cause of renal failure, duration of HD and previous treatment of pruritus was taken. Most of the patients received dialysis 3 times a week; only 8 of them received dialysis 2 times a week. The average dialysis vintage was 48.8 ± 51.9 months, and the hemodialysis was performed using bicarbonate dialysate. All individuals were undergoing hemodialysis using high-flux polysulfone membrane dialyzers. The single pool Kt/V accessing the dialysis adequacy for the whole study population was 1.28 (SD ± 0.39). In a group of patients reporting CKD-aP, it was 1.21 (SD ± 0.31), and in a group of patients not reporting CKD-aP, it was 1.34 (SD ± 0.43). The difference between these two groups was not statistically significant. Therefore, the level of hemodialysis adequacy did not have an impact on itch sensation.The most common cause of end-stage renal disease in the entire study cohort was glomerulonephritis (19.8%); in Group A it was diabetic kidney disease (17.7%) and in Group B it was also glomerulonephritis (26.6%). Substantively, the most frequent cause of kidney disease in the general population is diabetic nephropathy. However, currently published studies emphasize that the cause of kidney failure does not determine CKD-aP ,35.The average dialysis vintage was 48.8 ± 51.9 months. More than half of the HD patients underwent dialysis through an arterio-venous fistulae (65.1%) and the rest through a tunneled internal jugular central venous catheter. This research obtained ethical approval by the Wroclaw Medical University Ethics Committee . Patients were included in the research after obtaining their informed consent.Pruritus severity was assessed using the NRS, which is an easily accessible instrument commonly used to evaluate the intensity of pruritus. On a scale from 0 to 10 points, participants indicated the worst itch intensity they experienced during the last 3 days. Patients were classified according to the severity of pruritus as follows: NRS < 3—mild pruritus, NRS > 3 and < 7—moderate pruritus, NRS > 7 and < 9—severe pruritus and NRS > 9—very severe pruritus . Moreoveg rpm for 15 min. The received serum was stored at—80 °C until the next laboratory steps were performed. The serum level of IL-31 was subsequently measured by the ELISA (enzyme-linked immunosorbent assay) technique using the Nori Human IL-31 ELISA Kit , according to the manufacturer’s instructions. The absorbance of the sample was measured at 450 nm using the EPOCH  adjustable microplate reader. IL-31 had a test range of 50–3200 pg/mL and a sensitivity of 10 pg/mL.Blood samples of 9 mL were collected from all of the participants. Blood was drawn during the routine drawing-of-blood from an arterio-venous fistula or from a permanent HD catheter just before starting a dialysis session in dialysis patients. Subsequently, the samples were centrifuged at 3000× p of a value lower than 0.05 was considered significant.A statistical analysis of the obtained results was performed with the use of the IBM SPSS Statistics v. 26  software. All data were assessed for normal or abnormal distribution. The minimum, maximum, mean and standard deviation were calculated. Quantitative variables were evaluated using the Mann–Whitney U test and Spearman’s or Pearson’s correlations. For the qualitative data, the chi-squared test was used. For differences in more than two groups, the Kruskal–Wallis one-way analysis of variance on ranks was performed. A two-sided